The invention relates generally to receptor proteins and to DNA and RNA molecules encoding therefor. In particular, the invention relates to a variant human xcex17 subunit in which there is a substitution of the valine-274 position of the wild-type human xcex17 subunit. The invention also relates to DNA and RNA molecules that encode the variant human xcex17 subunit, as well as to methods of using the variant subunit to identify compounds that interact with it.
This background considers the variant xcex17 subunit as it relates to the nicotinic acetylcholine receptor (nAChR). The nAChR is comprised of transmembrane polypeptide subunits that form a cation-selective ion channel gated by acetylcholine (ACh) and other ligands. The hydrophobic transmembrane 2 (xe2x80x9cTM-2xe2x80x9d) region from each subunit is believed to form the wall of the ion channel.
Two of the more prominent nAChRs in brain are those containing xcex14 subunits and those containing xcex17 subunits (Sargent (1993) Annu. Rev. Neurosci. 16:403-443; Court et al. (1995) Alzheimer Disease and Associated Disorders 9:6-14). Mutations of the xcex14 and xcex17 subunits may underlie some diseases of the nervous system. For example, mutations of the xcex14 subunit have been associated with some forms of epilepsy (Beck et al. (1994) Neurobiol. Disease 1:95-99; Steinlein et al. (1995) Nature Genetics 11:201-203). Additionally, xcex17-containing nAChR may be involved in sensory processing related to schizophrenia (Freedman et al. (1995) Biol. Psych. 38:22-33; Rollins et al. (1995) Schizophr. Res. 15:183; Stevens et al. (1995) Psychopharmacol. 119:163-170), cytoprotection (Donnelly-Roberts et al. (1996) Brain Res. 719:36-44; Akaike et al. (1994) Brain Res. 644:181-187; Martin et al. (1994) Drug Dev. Res. 31:135-141; Quik et al. (1994) Brain Res. 655:161-167), and neurite growth and innervation (Chan et al. (1993) Neurosci. 56:441-451; Pugh et al. (1994) J. Neurosci. 14:889-896; Freeman (1977) Nature 269:218-222; Broide et al. (1995) Neurosci. 67:83-94).
A splice variant involving the TM-2 region of the xcex17 subunit has been detected in bovine chromaffin cells (Garcxc3xada-Guzmxc3xa1n et al. (1995) Eur. J. Neurosci. 7:647-655), and a naturally-occurring mutation of a protein homologous to the xcex17 subunit found in Caenorhabditis elegans, leads to neurodegeneration (Treinin et al. (1995) Neuron 14:871-877). The latter is a single amino acid mutation in the TM-2 region similar to the chick xcex17 valine-251 to threonine (xe2x80x9cc-xcex17V251Txe2x80x9d) mutation, one of several mutations artificially introduced into the chick xcex17 subunit to facilitate the study of xcex17 nAChR structure and subunit function (Bertrand et al. (1995) Sem. Neurosci. 7:75-90).
Compared to the chick xcex17 wild-type (xe2x80x9cc-xcex17WTxe2x80x9d) nAChR, c-xcex17V251T (also referred to as xcex17-4) retained high calcium permeability but desensitized slowly, and was 180-fold more sensitive to ACh. In addition, the c-xcex17V251T nAChR responded to dihydro-xcex2-erythroidine (xe2x80x9cDHxcex2Exe2x80x9d), normally an nAChR antagonist at xcex17 and other wild-type nAChR, as if it were an agonist (Galzi et al. (1992) Nature 359:500-505; Bertrand et al. (1993) Proc. Natl. Acad. Sci. USA 90:6971-6975). These studies have led to a model delineating the structure of the pore-lining TM-2 region, and the hypothesis that specific mutations within the TM-2 region can generate ligand-gated ion channels that conduct current in the receptor-desensitized state in addition to the normal receptor-activated state (Bertrand et al. (1995), supra; Bertrand et al. (1992) Proc. Natl. Acad. Sci. USA 89:1261-1265; Galzi et al. (1995) Neuropharmacol. 34:563-582).
Although the chick xcex17 nAChR is pharmacologically similar to the mammalian xcex17 nAChR, there are significant differences. For example, 1,1-dimethyl-4-phenylpiperazinium (xe2x80x9cDMPPxe2x80x9d) is a very weak partial agonist in the chick xcex17 nAChR, but is a highly efficacious agonist at the human xcex17 nAChR (Peng et al. (1994) Mol. Pharmacol. 45:546-554). Despite these differences, it would be expected that amino acid changes in the human xcex17 nAChR that are analogous to those in the chick xcex17 nAChR, particularly in critical TM-2 amino acids, would result in similar pharmacological and electrophysiological changes.
The present invention relates to a variant human xcex17 subunit in which valine-274 has been changed in analogy with the corresponding chick receptor variant. This variant is analogous to the chick xcex17V251T variant with regard to the relative position of the amino acid substitution in the TM-2 region. However, the variant human xcex17 subunit exhibits unexpectedly different pharmacological and electrophysiological characteristics.
The xcex17 subunit combines with itself and may combine with other subunits to create various nicotinic acetylcholine receptors. The possibility of combination with yet other proteins, which may or may not be identified as components of other classes of receptor, is not necessarily excluded.
Accordingly, in one embodiment, a DNA molecule or fragments thereof is provided, wherein the DNA molecule encodes a variant human (xcex17 subunit in which the valine-274 has been replaced.
In another embodiment, a recombinant vector comprising such a DNA molecule,or fragments thereof, is provided.
In another embodiment, the subject invention is directed to a variant human xcex17 subunit in which the valine-274 has been replaced.
In still other embodiments, the invention is directed to messenger RNA encoded by the DNA, recombinant host cells transformed or transfected with vectors comprising the DNA or fragments thereof and methods of producing recombinant polypeptides for the treatment of neurodegenerative processes, enzymatic function, affective disorders and immunofunction, using such cells.
In another embodiment, compounds such as antagonists are provided, as well as antisense polynucleotides, which are useful in treating conditions such as neurodegenerative processes, enzymatic function, affective disorders and immunofunction. Methods of treating individuals using these compounds and antisense polynucleotides also are provided.
In yet another embodiment, methods and reagents are provided for detecting the xcex17 variant.
In yet another embodiment, the invention is directed to a method of expressing the human xcex17 subunit variant in a cell to produce the resultant xcex17 variant.
In a further embodiment, the invention is directed to a method of identifying compounds that modulate the subunit or receptors containing the subunit and to a method of identifying cytoprotective or other therapeutic compounds using such cells.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.